PROJECT SUMMARY I am an instructor of Medicine at Johns Hopkins University and my goal is to become an independent physician-scientist in the field of heart regeneration with a focus on deciphering the mechanisms of stem cell regeneration therapy in damaged myocardium. Cardiac regeneration therapy holds great potential to repair damaged hearts and improve their function. In the past 15 years, several trials have tested the efficacy of various types of stem cell-based therapies in different cardiomyopathy models with varying results. A critical question that remains unanswered is whether stem cells and/or progenitor cells can differentiate into cardiomyocytes capable of establishing stable electromechanical integration with the host cardiac tissue to generate meaningful force and thus significantly improve systolic function. Alternatively, it is also possible that the beneficial effects are due to paracrine factor and exosome secretions that boost reparative pathways and prevent cell death. In this study, I propose a novel approach to directly test this fundamental question by using a conditional power switch to control the excitation and contraction, of stem cell-derived cardiomyocytes. Excitation and contraction will be controlled by conditionally expressing a GTPase named Rem1, which inhibits the voltage-activated calcium channel Cav1.2 (ICa,L), a channel essential for cardiomyocyte excitation. Rem1 under control of a doxycycline-inducible promoter was introduced into stem cells that differentiate into cardiomyocytes and upon doxycycline induction can be turned on or off. This proposal will introduce these myocytes into infarcted rat hearts and repetitively turn the power switch on and off to assess their systolic contributions. The influence of Cav1.2 inhibition by Rem1 on paracrine factor and exosome secretions, as well as transcription factor expression and mitochondria function of stem cell-derived cardiomyocytes will also be studied. Overall, this proposal will provide important insights into the mechanisms of cardiac regeneration therapy that will be critical for future refinement and widespread clinical application of this treatment. To successfully carry out this proposal, I have assembled an outstanding team. This includes leaders in cardiac physiology such as my mentor Dr. David Kass and my consultant Dr. Leslie Tung, my co-mentor Dr. Chulan Kwon an expert in heart development, and my consultants Drs. Charles Murry a world leader in cardiac regeneration and Dr. Patrick Cahan an expert in bioinformatics. They will all provide outstanding training in every method I need and will oversee and support my scientific progress and career development as an independent investigator. I already have my own lab space, lab technician and funds and I will have full access to state-of-art equipment both at the Johns Hopkins University Core facilities and in the division of Cardiology.